Polarization of renal tubular cells, by basolateral sorting of Na,K-ATPase, is crucial to cell volume regulation and vectorial transport of ions and nutrients. The mechanisms by which this polarized state is maintained remain puzzling. The objective of this proposal is to characterize the factors that maintain the polarized microdomain distribution of Na,K-ATPase in renal epithelial calls, subsequent to its basolateral sorting. The hypotheses are (a) erythrocyte-type ankyrin plays a crucial role in stabilizing Na,K-ATPase polarity, and (b) renal-specific ankyrins exist, which may also participate in this process. The longterm goal is to understand the response of ankyrin-Na,K-ATPase interactions during renal injury, and their role during recovery of the polarized state. The role of erythrocyte ankyrin in maintaining Na,K-ATPase polarity will be defined by (a) determining ankyrin binding sites on Na,K-ATPase, (b) constructing mutant Na,K-ATPase subunits which do not bind ankyrin, and (c) analyzing distribution and Triton extractability of Na,K-ATPase in MDCK cells grown in ouabain and transfected with ouabain-resistant wild-type or mutant Na,K-ATPase subunits. The role of renal-specific ankyrin isoforms in maintaining Na,K-ATPase polarity will be defined by (a) molecular cloning of cDNAs encoding renal ankyrin isoforms, using probes already available, (b) determining binding sites of renal ankyrins within Na,K-ATPase, and (c) analyzing distribution and Triton extractability of complexes containing renal-specific ankyrins and Na,K-ATPase, within MDCK cells transfected with wild-type ouabain-resistant Na,K-ATPase. The role of erythrocyte and renal ankyrins in the disruption of renal Na,K-ATPase polarity following ischemia and its return during recovery will be examined by (a) quantitating the biosynthetic response of ankyrin mRNA and protein expression in renal tubular cells following in vivo ischemia and during recovery, and (b) analyzing distribution and Triton extractability of Na,K-ATPase and ankyrin isoforms in ischemia and recovering renal tubular cells. These studies will delineate the specific role of "erythrocyte" and "renal" ankyrins in maintaining the topographic assembly of Na,K-ATPase, and provide new insights into the cellular mechanisms by which this polarity is disrupted after renal ischemia, and is restored during recovery.